To breach the human immune defenses, intracellular bacteria can enter, hide and replicate within the host immune cells as well as subvert their immunological functions. This work will characterize the molecular mechanisms that are responsible for this phenomenon, potentially leading to development of novel anti-microbial drug targets. The intracellular human pathogen Legionella pneumophila is the causative agent of the severe bacterial pneumonia known as Legionnaire's disease. Inhalation of aerosolized bacteria results in outbreaks of disease, which can be fatal in people with suppressed immune function. Legionella utilizes an arsenal of proteins injected in the host cell to assume control over various host processes and establish an intracellular replicative niche. The goal of this study is to elucidate the function(s) of a family of Legionella pneumophila proteins that are predicted to alter host cellular responses via subversion of the host ubiquitination regulatory networks. These effector proteins contain the eukaryotic F-box domain, which targets proteins for ubiquitination. Preliminary data showed that these F-box effectors are translocated into the cytosol of host cells during infection and associate with ubiquitinated proteins. Initially the goal of this study is to map the molecular determinant governing the interaction of the F-box effectors with ubiquitinated proteins and the SCF E3 ubiquitin ligase complex in mammalian cells utilizing immunofluorescence and immunoprecipitation analyses. In vitro ubiquitination assay will be used to determine the capacity of these bacterial F-box effectors to recruit and ubiquitinate target proteins utilizing host ubiquitin ligases. Tandem tag affinity purification and proteomics analysis of ubiquitinated proteins targeted for modification by the F- box effectors would identify potential host regulatory elements that are targeted by Legionella during infection. This study will use the clinical isolate strain Lp1 to create deletion mutant lacking the F-box effectors and investigate the contribution of these proteins to the capacity of Legionella to infect and replicate in host cells. The ability of these mutant strains to ubiquitinate target proteins identified by the proteomics screen will be assessed utilizing a cellular model of infection. This work will elucidate the molecular mechanism enabling the human pathogen Legionella pneumophila to subvert host cellular processes by taking control of the host ubiquitination machinery, potentially identifying common features of subversion utilized by other pathogens capable of exerting similar control, which can be targeted by pharmaceuticals to control or prevent infection.